Background
CAR-T Therapy
CAR-T (chimeric antigen receptor T cell) therapy is one of the most promising treatment for cancer, with multiple ongoing clinical
trials worldwide and 2 therapies already approved by the FDA(June et al., 2018) .
The therapy engineers endogenous T cells from the patients with synthetic receptors for tumor antigens, enabling them to target tumor
cells. The synthetic receptor, or chimeric antigen receptor (CAR), consists of an extracellular antibody-derived single chain
variable fragment (scFv) domain that can bind specific antigen with high affinity, a transmembrane domain, and an intracellular
signaling domain. The 1st generation CAR molecules only include a CD3zeta signaling domain, the minimal domain for T cell activation,
while 2nd and 3rd generation CARs also include one or more co-stimulating domains from CD28, CD137, 4-1BB, or others. The addition
signals enhance cytokines production and facilitate expansion of CAR T cells population upon repeated exposure to target
antigens(June et al., 2018) .
Similar to other types of adoptive cell transfer (ACT) therapy, CAR T therapy requires drawing peripheral blood from the patients,
Sorting out T cells, introducing new receptors by transducing T cells with lentiviral vectors, and expanding the population in vitro
before re-infusion into patients.
CAR T therapy has achieved unprecedented success in hematological cancer. In several clinical trials using CAR T to treat leukemia
and lymphoma, high percentage of patients experienced partial or complete response(June and Sadelain,
2018) .
The Adverse Effects
However, without proper control after administration of CAR-T cells, severe adverse effects associated with CAR-T therapy may bring
fatal risks to the patients, especially during the clinical trial stages. The most common side effects include cytokine release
syndrome, on-target off-tumor toxicity and off-target toxicity.
cytokine release syndrome (CRS)
CRS is considered the most common acute toxicity in the CAR T therapy, with patients experiencing fever, tachycardia, hypotension,
and other symptoms after infusion of CAR T cells. The cytokines can be produced by CAR T cells themselves or by other immune cells
like macrophages. Likewise, a wide range of cytokines participate in these pathological conditions, including interleukin-6 (IL-6),
IL-2 and interferon-γ(Maude et al., 2014) . The CRS development correlates with high level of
CAR T cells number and peak level of several cytokines and is also related to tumor burden(Brentjens
et al., 2013) .
Neurotoxicity
Neurotoxicity of CAR T therapy has been reported by several research groups. The symptoms include confusion, delirium, expressive
aphasia, obtundation, myoclonus, and seizure. Similar neurological toxicity has been reported with blinatumomab, a bispecific anti-
CD19 anti-CD3 antibody(Brudno and Kochenderfer, 2016) . However, the underlying mechanism
remains unknown(June et al., 2018) .
On-target off-tumor toxicity
On-target off-tumor toxicity is induced when the target antigen is also expressed on non-tumor tissues, despite its lower expression
level, and CAR T cells can inflict normal tissue damage by target antigen recognition. Such toxicity has been documented in the
literature and has even lead to fatality. In one study 3 patients with metastatic renal cell carcinoma receiving CAR T cells
targeting carboxyanhydrase-IX (CAIX) developed liver damage, due to unexpected expression of CAIX on the bile ducts
(Lamers et al., 2013) . The risk is further exemplified by another study in which a patient
with metastatic colorectal cancer experienced acute respiratory distress and pulmonary edema and subsequently died after infusion of
CAR Ts targeting ERBB2 (Her-2/neu), possibly due to expression of ERBB2 on lung tissue(Morgan et al.,
2010) .
Cross-reactivity
Cross-reactivity occurs when CAR T cells recognize antigens on normal tissues similar to target antigen, possibly leading to organ
damage. By far no cross reactivity of CAR T cells has been reported, instead such toxicity has been observed in clinical trials using
modified TCR T cells(Cameron et al., 2013) .
Current Methods
Immunosuppressive drugs
To date the most common method for CRS management is giving immunosuppressive drugs. Tocilizumab (Actemra), an anti-interleukin-6-
receptor antagonist originally developed for rheumatoid arthritis, is often effective in the management of CAR T cells related
cytokine release syndrome and has been approved by the FDA for such treatments(Brudno and
Kochenderfer, 2016) .
Corticosteroids are given when patients are not responsive to Tocilizumab, but they are not considered as the first option since they
can interfere with T cell functions and induce T cell apoptosis(Brudno and Kochenderfer, 2016)
, with some research demonstrating that prolonged administration of corticosteroids can impair CAR T therapy efficacy
(Davila et al., 2014) .
A potential risk for immunosuppressive treatment is its global effect on the immune system, rendering the patients more vulnerable to
opportunistic infections by bacteria, fungi and viruses. It has been demonstrated that rheumatoid arthritis patients have higher rate
of respiratory infections(Hoshi et al., 2012) . In addition, a recent study suggested a
correlation between CRS and rate of infection(Hill et al., 2018) . Although no causative
conclusion can be drawn that the immunosuppressive treatment for CRS leads to those infection, the risk of immunosuppression-
associated infections should not be overlooked, and it is desirable to mitigate adverse effects in CAR T therapies without global
inhibition on the immune system.
Suicide Genes
Compared to the systemic effect of immunosuppressive drugs, genetic modification of the infused CAR T cells is able to provide a more
precise control on the infused cell population alone. A straightforward method is to engineer suicide genes in CAR T cells and remove
transferred cell population when giving drugs(Marin et al., 2012) . The first suicide gene
evaluated in the clinical trials is thymidine kinase from herpes simplex virus, which can induce cell death by DNA synthesis
inhibition upon treatment with ganciclovir. Inducible dimerization of Fas or caspase 9 serves as a second strategy for selective
depletion of modified cells. The inducible caspase 9 (ICas9) molecule contains an FK506 binding domain and is able to mediate
dimerization of caspase 9 domain when adding AP1903, and ultimately induce cell apoptosis(Budde et
al., 2013) . A third method is achieved by programming the cells to express truncated epidermal growth factor receptors on the
cell surface, which can be targeted by antibody and induce antibody-dependent cell-mediated cytotoxicity (ADCC)
(Wang et al., 2011) .
However, these methods could deplete CAR-T cells and halt the expensive treatment. Repeat the treatment may be a burden on the
patients, both physically and financially.
Controllable Receptors
Another strategy is to engineer the CAR receptors, rendering them requiring additional signals, typically a small molecule drug, to
fully activate. Wu et al. (Wu et al., 2015) first demonstrated the such strategy by
constructing a split synthetic receptor, with one chain containing the extracellular scFv, hinge and costimulatory domain and the
other containing the activation domain of CD3zeta. When adding small molecule Rapalog, the heterodimerization of 2 chains
reconstitutes a full CAR receptor and therefore can generate full activation signals. Similarly, Juillerat et al.
(Juillerat et al., 2016) modified the hinge domain to include a FKBP/FRB dimerization pair,
which separate the scFv from the cell membrane. When AP21967 (a rapamycin analog) is added, the interaction of FKBP and FRB fold the
extracellular domain into an optimal conformation for activation.
However, modification of receptors may be troublesome that it may alter the structure and conformation of the receptors, which in
turns may interfere with CARs' function. Such notion is exemplified in the research of Wu et al., that different split methods
resulted in different signaling outcomes, with most of the receptor pairs failing to fully activate in the presence of drugs
(Wu et al., 2015) . Considering a lack of conclusive information on mechanisms of CAR T cell
activation(Harris and Kranz, 2016) and the diversity of existing CARs in clinical
research(June et al., 2018) , a method must be developed for each type of CAR to ensure it
works as expected, which can be labor-intensive.
Furthermore, such methods are limited to CAR Ts alone, which have synthetic receptors. In contrast, TCR Ts rely on the endogenous T
cell receptor complex to function properly, and TCRs, as part of the complex, requires native conformation to function, leaving
little, if any, space for modification.
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